to Introduction Parasite diversity labs Overview The objective of this segment of the course is to introduce you to the diverse life histories and extensive adaptive radiation of animal parasites. This is accomplished through the combined use of a standard parasitology slide box, Internet material and specific demonstration materials. You are encouraged to work at your own pace with the slide boxes, using this lab manual, plus supplementary texts and diagrams as a guide. The latter will be available to you in the lab. For those of you who wish to be more independent, I urge you to visit at least one of these excellent web sites: http://asp.unl.edu/photo.html http://www.biosci.ohio-state.edu/~parasite/home.html Both are award-winning sites that detail virtually all of the parasites that we will cover in this section of the course. The slide box is a valuable tool for appreciating the functional morphology and diverse life cycles of common groups of animal parasites. It is here where you will be exposed to parasite adaptations and diversity. I have only included the bare minimum of slide material. However, thorough familiarity of the slides in your slide box will be an essential part of the course. You are responsible for understanding and properly using the terminology found within this manual. Most terms are defined in the text descriptions, in my lectures, or in the material available on lab day. Study the slides carefully and compare specimens to the diagrams and descriptions provided in this manual and in the texts available. Make sure that you make labeled drawings for your future reference for each of the specimens. Your book will be an invaluable reference source as you go through your slide box. You will get the greatest benefit from the slide box if you try to integrate the morphology of each species with its life-cycle and by identifying similarities and differences among related species. Where does the parasite live in the host ? How does it attach ? How does it feed? How does it reproduce and transmit between hosts ? Morphology can provide many clues to the life cycle of a parasite and life cycles can suggest the types of morphological adaptations that might be necessary.
Classification scheme for species referred to in this manual The classification scheme used in this manual is greatly simplified. However, I do ask that you become familiar with the names of the few species I have included (to genus). All of the following species are in your slide box or will be available on demonstration.
Other species may also be presented as demonstration material, depending on their availability. Microparasites: The Protozoa (note: the phylogeny of this group is extremely contentious. Each of the texts in the lab treats the subgroups differently. I use a fairly traditionaly scheme here. You are not responsible for the detailed phylogeny outlined here I provide it for convenience only). Phylum Sarcomastogophora Class Zoomastigophora Order Kinetoplastida Trypanosoma rhodiense and T. brucei Order Trichomonadida Trichomonas vaginalis Order Diplomonida Giardia lamblia Phylum Apicomplexa Plasmodium falciparum Cryptosporidium parvum Toxoplasma gondi Macroparasites: Phylum Platyhelminthes Class Digenea Order Strigeata Schistosoma mansoni Ornithodiplostomum ptychocheilus Posthodiplostomum minimum Order Echinostomata Echinostoma revolutum Fasciola hepatica Class Cestoidea Diphyllobothrium dendriticum Triaenophorus crassus Echinococcus granulosis Phylum Acanthocephala Neochinorhynchus sp. Polymorphus paradoxus Phylum Nematoda
Trichinella spiralis Trichuris trichura Necator americanus (or Ancylostoma caninum) Wucheria spp. Phylum Arthropoda Class Crustacea Argulus Sacculina Hemobaphes Class Arachnida Argas persius Dermacentor andersoni Class Insecta Diamanus Pediculus humanis
Parasite diversity I. Microparasites The microparasites include the protozoans, bacteria, fungi and viruses. They are characterized by their small size (almost always microscopic and often only 1-2 micrometers in size), their modes of reproduction, and their ability to illicit strong and specific host immunity. They are fascinating to study, partly because they include the majority of the major parasitic diseases of humans, but also because they demonstrate brilliant adaptations for the parasitic way of life. Unfortunately, their small size makes them exceptionally difficult to study. This difficulty leads to the fact that we know so little about the general biology of even our most pathogenic microparasites. In this lab, living material is very difficult to acquire. We will focus on the protozoan parasites via demo material, slide material and text material. The parasitic Protists The study of protozoans requires considerable patience and skill as a microscopist. In order to find the diagnostic characters on your slides, you will probably require the use of oil immersion techniques (if you are shaky on the use of this technique, ask me for a refresher!). The parasitic Protists are an extremely diverse assemblage of species. Your slide box contains only a very small sample of this diversity. As you go through the protozoans, pay particular attention to the many morphological differences between species, as well as the diversity of life-cycles. You will see that there are direct and indirect modes of transmission, sexual and asexual multiplicative phases and various levels of response by the host. Rather than provide detailed line figures of the various types of parasitic protists, you should use the material available to you (texts, websites) to draw your own! In a nutshell, you should be able to sketch the life-cycles of each of the groups listed below, and you should be able to make a rough sketch of the generalized body plan. Phylum Mastigophora Trypanosoma rhodiense and T. brucei T. rhodesiense is the causative agent of the dangerous human disease, African sleeping sickness. Over 10,000 cases are diagnosed annually; 50% of these die and the other 50% suffer permanent damage. T. brucei is responsible for the immensely important disease of livestock known as nagana. Both species use tsetse flies as vectors. African sleeping sickness has plagued humans ever since they first encroached on the domain of the tsetse fly. The disease has kept over 4 million sq miles of grazing land in Africa out of agricultural production. Consider the political, ecological, and socioeconomic implications if a vaccine for sleeping sickness were ever found (note: a third species, T. cruzi, is known as American trypanosomiasis and infects over 10 million people; it uses a reduvid bug as vector).
This group is characterized by a spindle-shaped body containing a central nucleus (or kinetosome). The flagellum arises from this structure. A kinetoplast is also situated near the base of the flagellum. It is a mass of DNA within a single mitochondrion and is easily seen with the light microscope. Your slide is a smear from an infected humans blood. You will see numerous trypomastigotes. This stage is the final developmental stage of the trypansomes (see diagram in text or website). Under oil immersion, you should be able to see the undulating membrane, flagellum, nucleus and kinetoplast. Trypomastigotes undergo asexual reproduction (binary fission) in the vertebrate host (human, domestical mammal, native ungulates). The fact that the parasite can survive in so many reservior hosts is a major factor in the epidemiology of the disease. During a blood meal, the tsetse flies ingest infective trypomastigotes. Once ingested by the fly, these multiply by binary fission and then metamorphose to a long and thin form within the insects forgut. These migrate to the salivary gland and transform into a further form, which can then be inoculated into another mammalian host during a blood meal. This species is fascinating for its ability to evade the hosts immune system by continuously changing the antigenic structure of their external surface (the phenomenon of antigenic variation). Trichomonas vaginalis This is a cosmopolitan species, found in the urogenital tracts of men and women. You are not responsible for the functional morphology of this group. I include it for two reasons. First, this species is one of several parasitic protists that are transmitted via direct physical contact (T. vaginalis is primarily an STD). It therefore represents a third type of transmission strategy (in addition to using a vector and via cysts). Members of this family span the range of symbiotic life-styles, from purely free-living, to commensal (one group is found only in the gut of termites; another is found between the teeth, and on the tonsils, of humans) to parasitic. Refer back to your notes on the relationships between Rhizobium and Agrobacterium, and between the pathogenic vs. mycorrhizal fungi. Can you imagine any parallels ? Members of this group are easily recognized by having an anterior tuft of flagella, a stout median rod and an undulating membrane. Can you imagine the selection pressures occurring on the Trichomonads that may have led to their different ways of exploiting hosts ? T. vaginalis is transmitted primarily through sexual intercourse. Many strains are of very low pathogenicity, especially in men. Some strains, particularly when infecting women, can cause an intense inflammation, with itching and copious white discharge that is swarming with trichomonads. They feed on bacteria, leukocytes and cellular debris. Giardia lamblia Your slides contain the feeding form (trophozoite) and cysts of this, the most common intestinal parasite of humans. It is the causative agent of giardiasis or beaver-fever. This is the disease most associated with wilderness campers and is typically associated with severe diarrhea. The parasite matures in many reservoir hosts (such as beavers and cattle) which is an important in factor the epidemiology of this disease.
Giardia has 4 pairs of flagella arising from the centre of the cell; they are typically lost during staining. Using the oil immersion lens you should be able to see two nuclei and a central pair of median bodies. The trophozoites are cup-shaped and the surface of the ventral side is concave and thickened to form a large adhesive disk, used for attachment to the hosts intestinal villus (see diagram in text or website). Locate a cyst using the high power objective and advance to oil immersion power and note the thick cyst wall, enclosed flagella, nuclei and median bodies. Mammalian definitive hosts ingest infective cysts in drinking water. Giardia excysts in the intestine, releasing trophozoites. These attach to villi and multiply by binary fission. In victims of giardiasis massive infection is typical; the presence of several billion cysts in a single stool is not unusual! Cysts are the infective, resistant stage. They can withstand freezing and stomach acidity. Unlike the trypansomes, only one host is required to complete the life cycle. Phylum Apicomplexa All members of this phylum are parasitic (recall that it is quite unusual for any phyla to be exclusively parasitic) and they infect members of all animal phyla. All are characterized by an apical complex that is only revealed under the electron microscope. This structure is important for recognition and penetration of host cells. Members of the phylum include some of the most serious diseases of humans and domestic animals. The life cycles of apicomplexans are complex; with direct or indirect pathways. Species with direct life cycles have resistant spores or oocysts that bridge the gap in the external environment between hosts. Those with an indirect life cycle involving vectors remain within a host and thus have no need for a protective cyst. Monocystis lumbrici is an Apicomplexan parasite that lives in the seminal vesicles of terrestrial earthworms. The worm becomes infected when it ingests a spore containing several sporozoites. These hatch in the gizzard, where the released sporozoites penetrate the intestinal wall, enter the dorsal blood vessel, and then make their way to one of the hosts 5 or so hearts. From there they penetrate the seminal vesicle, where they enter the sperm-forming cells in the wall. At this point they ingest and destroy the developing spermocytes. Then they move into the lumen of the vesicle where they become mature trophozoites. After a period of feeding, two of these will come together, flatten against each other, and secrete a common cyst around each other. This is the gametocyst, usually containing 2 gamonts. Each now undergoes extensive division of their nuclei, pinches off a small portion of cell cytoplasm, which together then bud off to become the gametes. The fusion of a pair of gametes forms a zygote, each ultimately becoming a spore. Three cell divisions later forms 8 sporozoites. Thus, each gametocyst now contains many oocysts. New hosts become infected by ingesting gametocysts, or more commonly, by ingesting individual oocysts. Thus, meiosis is zygotic. Only the zygote is diploid, and reductional division in sporogony returns the sporozoites to the haploid condition.
Proceedure: Dissect the anterior end of a freshly anesthetized worm. Remove the seminal vesicles and place in a drop of water. Take small pieces of seminal vesicle, squash under a cover slip and look for the different stages of Monocystis (see figure above). Make drawings of each stage and construct an annotated life-cycle. Record as many different stages of infection as you can. Plasmodium falciparum This intracellular parasite is the most dangerous of the 4 species that cause malaria in humans. This species will be the focus of our discussions in lecture. It is the most common and debilitating of human parasitic diseases. Over two million people each year die from the disease. Moreover, the disease has played a major role in shaping our history and civilizations. It is impossible for you to understand this disease without a thorough understanding of its life cycle. Your slides only show a fraction of the various stages of the malaria life-cycle. One slide shows gametocytes inside host red-blood cells as deeper-staining structures (often
crescent or bean-shaped). Using the oil immersion lens you should see that microgametocytes have a large nucleus and irregularly distributed granules. Macrogametocytes have a small compact nucleus with a dark red nucleolus. Further devleopment of these forms only continues inside a mosquitos stomach. Sometimes, the gametocyte-infected cells have been distorted to such an extent that it ruptures during the fixing process. Thus, you may see some gametocytes which are not enclosed within a red blood cell membrane. You also have slides which show trophozoites. They can be distinguished by the large food vacuole surrounded by a thin layer of cytoplasm and including a peripheral nucleus. This gives the characteristic ring which forms the well-known ring-stage. Again, the infected RBC may appear distended or abnormal in shape. Use the demonstration slides of the various life-cycle stages and life-cycle diagrams to help you understand the biology of this important parasite. Cryptosporidium parvum This waterborn parasite, together with Giardia, represents one of the two major waterborn parasites of humans. Although the genus was first discovered many years ago (as a parasite of turkeys), it has only recently received attention from biologists and disease specialists. In 1982 the American Centre for Disease Control, found 21 males from large cities to be suffering from severe diarrhea; all had AIDS. Then, in 1993, there was a severe outbreak of cryptosporidiosis in Milwaukee, affecting over 400,000 people. There has since been an outbreak in Kelowna (1995), in Shaughnessy (near Picture Butte), Alberta in 19...